The present invention relates to the detection and treatment of heart failure based on the down-regulatory activity of the AUF1 or an AUF1-related polypeptide toward human xcex2-adrenergic receptors.
The condition of heart failure is associated with heightened activity of the adrenergic nervous system (Bristow et al., Circulation 82:(Suppl.I) 12-25 (1990)), the severity of failure correlating with increases in circulating and cardiac concentrations of the catecholamine, norepinephrine (Cohn et al., N. Engl. J. Med. 311:819-823 (1984)). As a consequence of this increased xe2x80x9cadrenergic drivexe2x80x9d the cardiac xcex2-adrenergic receptor (xcex2-AR)/G-protein/adenylyl cyclase pathway can become markedly desensitized. One major component of the desensitization is selective down-regulation of the dominant adrenergic receptor subtype within the human myocardium, the xcex21-AR (Bristow et al., Circulation 82:(Suppl.I) 12-25 (1990); Bristow et al., Circ. Res. 59:297-309 (1986); Brodde et al., J. Cardiovasc. Pharmacol. 8:1235-1242 (1986); Bristow et al., J. Clin. Invest. 89:803-815 (1992)). Recently, it has been demonstrated that the observed decrease in xcex21-adrenergic receptors in failing human heart is closely associated with a corresponding down-regulation of xcex21-AR mRNA (Bristow et al., J. Clin. Invest. 92:2737-2745 (1993); Ungerer et al., Circulation 87:454-463 (1993)).
Experiments performed using hamster DDT1-MF2 smooth muscle cells (Hadcock et al., J. Biol. Chem. 264:19928-19933 (1989)) suggest that down-regulation of the endogenously expressed xcex22-AR mRNA does not appear to be caused by a decrease in the rate of transcription; rather, it appears that agonist exposure decreases the half-life of xcex2-AR mRNA from approximately 12 to 5 h (Hadcock et al., J. Biol. Chem. 264:19928-19933 (1989)). This regulatory mechanism has been demonstrated previously to be important for numerous mRNAs encoding proto-oncogenes, lymphokines and cytokines. For these gene products regulation of mRNA stability has also been associated with the interaction of the mRNA with a family of cytosolic proteins (Mr 30,000-40,000) that often bind to A+U-rich elements (ARE) commonly within the 3xe2x80x2 untranslated region (3xe2x80x2UTR) of the mRNA. This interaction induces mRNA degradation by mechanisms poorly understood. However, for some mRNAs including those containing AREs (Savant-Bhonsale and Cleveland, Genes Dev. 6:1927-1939 (1992); Winstall et al., Mol. Cell. Biol. 15:3796-3804 (1995)), the degradation of mRNA may be associated with the process of translation. The cytosolic A+U-rich mRNA binding proteins are in general considered to be distinct from other mRNA binding proteins such as the heterogeneous nuclear ribonucleoproteins (hnRNPs) (Dreyfuss, Annu. Rev. Cell Biol. 2:459-498 (1986); McCarthy and Kollmus, TIBS 20:191-197 (1995)), however, the role of hnRNP A1 and C proteins as cytoplasmic factors regulating mRNA stability is currently undergoing reassessment (Gorlach et al., EMBO J. 11:3289-3295 (1992); Hamilton et al., J. Biol. Chem. 268:8881-8887 (1993)).
From previous studies (Port et al., J. Biol. Chem. 267:24103-24108 (1992); Huang et al., J. Biol. Chem. 268:25769-25775 (1993); Tholanikunnel et al., J. Biol. Chem. 270:12787-12793 (1995)) using cytosolic extracts produced from DDT1-MF2 hamster smooth muscle cells, the properties of a xcex2-AR mRNA-binding polypeptide (xcex2-ARB), which binds to hamster xcex22-adrenergic and human xcex21-adrenergic receptor mRNAs, have undergone preliminary characterization. Binding of xcex2-ARB to mRNA was determined to involve regions of the 3xe2x80x2UTR of the hamster xcex22-AR mRNA containing an ARE (Port et al., J. Biol. Chem. 267:24103-24108 (1992); Huang et al., J. Biol. Chem. 268:25769-25775 (1993)). In addition, agonist stimulation of the xcex2-AR pathway or protein kinase A (PK-A) activation by a cAMP analogue resulted in significant up-regulation (3-4 fold) of xcex2-ARB as detected by UV-crosslinking. Conversely, treatment of DDT1-MF2 cells with dexamethasone, which up-regulates xcex22-AR mRNA, down-regulated xcex2-ARB by xcx9c50%. Therefore, agents that regulate hamster xcex22-AR mRNA stability and abundance appear to affect reciprocally the abundance of xcex2-ARB. Among the family of G-protein coupled receptors, the mRNAs of the hamster xcex22-AR, the human xcex21- and xcex22-AR, and the thrombin receptor have all been demonstrated to interact with xcex2-ARB (Port et al., J. Biol. Chem. 267:24103-24108 (1992); Huang et al., J. Biol. Chem. 268:25769-25775 (1993); Tholanikunnel et al., J. Biol. Chem. 270:12787-12793 (1995)). To date, the identity of xcex2-ARB has remained unresolved.
The cytoplasmic RNA-binding polypeptide, AUF1 (A+U-rich element RNA-binding/degradation Factor) (Zhang et al., Mol. Cell. Biol 13:7652-7665 (1993)), has recently been cloned and characterized. AUF1 binds to the 3xe2x80x2UTRs of several highly regulated mRNAs including c-myc, granulocyte/macrophage colony-stimulating factor (GM-CSF), and c-fos. Further, there is evidence of xe2x80x9ccause and effectxe2x80x9d between AUF1 and regulation of mRNA stability in that partially purified AUF1 can selectively accelerate the degradation of c-myc mRNA in an in vitro mRNA decay system (Brewer, Mol. Cell. Biol. 11:2460-2466 (1991)).
The present invention involves a method of detecting in a biological sample the amount of the AUF1 or an AUF1-related polypeptide for indicating heart failure. This involves generating an antibody to the products of the AUF1 or an AUF1-related polypeptide. Then contacting the antibody with the biological sample and detecting the amount of immune complex formation as an indication of the amount of the polypeptide in the biological sample. A higher amount of immune complex formulation, than found in a normal cell, indicates that heart failure is present. The higher than normal amount of the AUF1 or an AUF1-related polypeptide is a result of a high level of the AUF1 or an AUF1-related gene. This higher than normal amount of AUF1 or AUF1-related polypeptide is involved in the compensatory response to heart failure and/or involved in the worsening the condition of heart failure. This detection procedure would preferably involve contacting a biological sample from a human patient with an antibody (e.g., monoclonal antibody) which specifically reacts with the polypeptide. Then determining the amount of immune complex formation present in the biological sample. A higher amount of immune complex formation, than found in a normal cell, indicates that heart failure is present. The biological sample can be obtained from human ventricular myocardium, serum, and blood cells.
Another method for determining heart failure is the use of probes that bind to the RNA of the AUF1 or an AUF1-related gene. This involves hybridizing an RNA extracted from a biological sample of a human patient with a probe specific for the gene. Then determining the degree of hybridization to the mRNA as an indication of the amount of the gene in the biological sample. A higher degree of hybridization, than found in a normal cell, indicates that heart failure is present.
Another aspect of the invention involves a method for detecting a pharmacological that inhibits the down-regulatory activity of the AUF1 or an AUF1-related polypeptide for limiting the expression of xcex2-adrenergic receptor genes by decreasing the binding affinity of the polypeptide for the xcex2-adrenergic receptor gene. This involves mixing the polypeptide, a radiolabeled ARE-RNA of the xcex2-adrenergic gene, and the pharmacological. The resulting mixture is filtered through a membrane such as nitrocellulose. Pharmacologicals that inhibits the down-regulatory activity of the polypeptide do so by decreasing the binding affinity of the polypeptide for the ARE-RNA. Since the polypeptide binds to the membrane, the radiolabeled ARE-RNA complexed with the polypeptide will adhere by means of the polypeptide. Any decreased binding affinity is reflected by the decreased counts per minute (cpm) retained on the membrane reflecting the decreased amount of bound radiolabeled ARE-RNA as a result of the presence of the pharmacological.
An additional aspect of the invention provides a composition containing a pharmacological in a pharmacologically acceptable carrier, suitable for treating a human having a high level of the AUF1 or an AUF1-related gene. These pharmacologicals can include small organic molecules, peptides, antisense RNA to AUF1 or AUF1-related mRNA, and a mini-peptide inhibitor/competitor to AUF1 or AUF1-related polypeptide.